Our description of the UGT1 locus has proven critical for the determination of genetic defects in patients with Crigler-Najjar(CN) diseases. The original version of the gene complex (UGT1A-UGT1F) codes for at least 2 bilirubin (Br), 3 Br-like, and 1 phenol transferases. In the 5' region, six different exons 1, each with an upstream promoter and each encoding the amino terminus of an isoform, are arrayed in series with 4 common exons encoding six identical carboxyl termini in the 3- region of the locus. The most 5'exon was used to select other cosmid clones to extend the locus by more than 125 kb. At least five additional exons 1 in three cosmid clones have been identified, mapped for restriction endonucleases, and analyzed by Southern hybridization. Two of the exons have been sequenced and arranged in the series. The newly identified exons are related to the unique region of a second phenol, HLUG P4, reported in the literature. We have isolated and sequenced the corresponding cDNA for one of the newly characterized exons 1, and the cDNA has been expressed in order to determine substrate specificity. Predictably, a critical mutation in a unique exon affects a single form, whereas one in a common exon inactivates the entire locus. Both the deletion of a Phe-170 and a G276R missense mutation in exon 1A of the UGT1A gene of separate CN Type I patients affect the amino terminus of the HUG-Br1 isoform. The code in patient 1 abolished a critical di- phenylalanine in a conserved hydrophobic Micro-region A (MR A, codons 161-180) which according to computer-determined secondary structure is critical to the formation of a buried hydrophobic helical structure, a structure not seen in all other isoforms (which lack the di- phenylalanine) examined. Also two conserved Pro in the MR A are required for activity. All mutations in MR A generated pH-sensitive activity; there was complete loss of activity at the newly uncovered major pH optimum of 6.4, but normal activity at the routinely used pH 7.6 assay condition. In patient 2 a missense mutation, G276R, disrupted a strictly conserved di-glycine (codons 276/277) in a conserved Micro-region B (MR B, codons 270-288). Mutations in MR B indicate that of two conserved Pro, the 170, but not 287, is required for activity; also conserved Val-275 is not required. For MR B loss of activity occurred at both pH 6.4 and 7.6. Evidence shows that the di-glycine is contained in a --hair pin loop and is present in all transferase isoforms despite its coding by a unique exon 1 at the UGT1 locus.